Process for production of peptide thioester

ABSTRACT

A process for chemically converting a peptide chain into a peptide thioester includes, when a —C(═X)—R 1  group is introduced to the thiol group of the cysteine residue and then the resulting peptide is reacted with a compound having a leaving group represented by the formula: —NH—C(═Y)NHR 3  in an organic solvent, the —NH—C(═Y)NHR 3  group binds via addition reaction to the carboxyl group of the N-terminal-side peptide bond of the cysteine residue, whereby the peptide bond is cleaved and the C-terminal-side peptide fragment is cut off. Further, when the resulting peptide chain having the —NH—C(═Y)NHR 3  group is reacted with a thiol in a buffer solution, a thiol exchange reaction occurs, namely, the thiol group of the thiol binds to the carbonyl carbon to which the —NH—C(═Y)NHR 3  group has bound, whereby the —NH—C(═Y)NHR 3  group is eliminated.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional Application of the Non-provisionalApplication No. 13/379,832, filed on Dec. 21, 2011, which is a nationalstage application of PCT/JP2010/060443 filed on Jun. 21, 2010, whichclaims priority to Japanese Application No. 2009-151713, filed on Jun.26, 2009. The present application claims priorities to all these priorapplications and incorporates these prior applications by reference intheir entirety.

TECHNICAL FIELD

The present invention relates to a process for production of a peptidethioester.

BACKGROUND ART

Various methods such as biosynthesis, chemical synthesis and cell-freesynthesis are known to be used for synthesizing proteins. In thebiosynthesis method, a protein is obtained by utilizing the inside of acell such as an Escherichia coli cell and introducing and expressing DNAencoding the protein to be synthesized in the cell. In the chemicalsynthesis, the objective protein is synthesized by sequentially bindingamino acids in an organochemical manner. In the cell-free synthesis, theprotein is synthesized in a cell-free system utilizing an enzyme, etc.,present in various cells such as the Escherichia coli cell. Thesemethods are appropriately used separately or combined depending on theintended use, the size and the nature to be added, of the protein.

At present, in order to synthesize a protein homogeneously having aparticular modification with a sugar chain or a lipid, etc., in a middlepart of its amino acid sequence, amino acids are modified with the sugarchain or the lipid, etc., in advance and then a peptide chain includingthe modified amino acids is chemically synthesized.

A solid-phase synthesis is mainly used as the method for chemicallysynthesizing the peptide chain. However, the peptide chain obtained bythe solid-phase synthesis is generally a short chain, and is composed ofat longest about 50 residues.

Thus, the short peptide chains are separately prepared and then they areligated in order to synthesize the long peptide chain having themodification. Various techniques for ligating the peptide chains havebeen reported, and one widely used technique is the native chemicalligation method (NCL method). The NCL method can also be applied betweenunprotected peptide chains, and is known to be useful for forming anative amide bond (peptide bond) at a ligation site (e.g., PatentLiterature 1). The NCL method is a chemoselective reaction between afirst peptide having an α-carboxythioester moiety at its C-terminal anda second peptide having a cysteine residue at its N-terminal, and athiol group (SH group, also referred to as a sulfhydryl group) of thecysteine side chain is selectively reacted with carbonyl carbon of athioester group, whereby a thioester binding initial intermediate isformed by the thiol exchange reaction. This intermediate spontaneouslyperforms intramolecular transposition to give the native amide bond atthe ligation site while it regenerates the cysteine side chain thiol.

In this method, two peptide chains can be ligated via the peptide bondonly by mixing the unprotected peptides in a buffer solution. In the NCLmethod, even when compounds such as peptides having many functionalgroups are reacted, the C-terminal of one peptide can be ligatedselectively to the N-terminal of the other peptide. From these points,it is important to determine in what way to utilize the NCL method inorder to chemically synthesize the protein.

However, a problem when the NCL method is utilized includes thepreparation of a peptide thioester having an α-carboxythioester moietyat its C-terminal, which is required as a raw material. Various methodshave been reported for preparing the peptide thioester, and thosemethods can be generally classified into two types based on thesolid-phase synthesis.

A first one is the method of constructing the peptide thioester on aresin. In this method, the peptide thioester can be obtained togetherwith cleavage of the peptide chain from the resin after constructing thepeptide (e.g., Boc solid-phase synthesis, Fmoc solid-phase synthesis). Asecond one is the method of constructing the peptide chain on the solidphase via a linker equivalent to thioester (Safety catch linker, Fujiimethod, Dawson method, Mercapto propanol method, Kawakami method,Danishefsky method, Hojo method, Aimoto method, etc.). In this method,thioester is obtained by activating the peptide chain C-terminalconstructed by appropriately treating with the linker, followed bythiolysis of the peptide chain (Non-patent Literature 1).

In addition to these methods, the method in which a protected peptide sothat the side chain is protected by the solid-phase synthesis and onlythe carboxyl group at the C-terminal is free is synthesized followed bythioesterification under an appropriate condensation condition has alsobeen reported (e.g., Patent Literature 2). Any of these methods havebeen well-established, and used for various protein syntheses. However,the size of the peptide thioester capable of being synthesized islimited because these methods are limited to restrictions of thesolid-phase synthesis. Further, in the method using the linker, anon-native amino acid derivative or a specific derivative must bechemically synthesized separately. Thus, their procedures cannot alwaysbe said to be simple.

An intein method solved the restriction of the thioesterification by thesolid-phase synthesis (Non-patent Literature 2). In this method, apolypeptide fragment biosynthesized from a cell can be obtained asthioester. In the intein method, the peptide chain is thioesterified byutilizing a protein splicing function that occurs in the particularprotein sequence, and the polypeptide chain is obtained as thioester. Anadvantage of this method is that a long chain peptide thioester can beobtained. The synthesis of the large modified protein, which had beenconsidered to be difficult to synthesize until now, has become possibleby combining this method with the chemical synthesis method (Non-patentLiterature 3). The method of expressing the polypeptide chain andobtaining it has been studied extensively, and well-established as abasic technique in biology.

However, when the intein method is used, a peptide sequence to betargeted is necessary and an expressed intein complex protein must befolded to take on an inherent three-dimensional structure because notonly is the polypeptide expressed but also the protein splicing iscaused to function. Thus, depending on the polypeptide sequence to beexpressed, the peptide thioester is not always obtained in associationwith sufficient conditions for optimization and the accompanyingcomplications in the work.

Meanwhile, the method of cleaving the peptide chain at a position of acysteine residue by reacting a compound with the SH group of thecysteine residue in the peptide (Non-patent Literatures 4 and 5), andthe method of cleaving the peptide bound to the solid phase using thelinker (Non-patent Literatures 6 and 7) are known as the methods ofcleaving the peptide. Also, the method of cleaving the peptide bond onthe C-terminal-side of a methionine residue using cyanogen bromide(CNBr) is known. However, these are not methods for obtaining a peptidefragment as the thioester.

RELATED ART LITERATURE Patent Literature

Patent Literature 1: International Publication WO 96/34878

Patent Literature 2: International Publication WO 2007/114454

Non-patent Literature

Non-patent Literature 1: Ingenito et al., J. Am. Chem. Soc., 121:11369-11374, 1999.

Non-patent Literature 2: Schwartz et al., CHEM COMMUN., 2087-2090, 2003.

Non-patent Literature 3: Muir, Annu. Rev. Biochem., 72: 249-289, 2003.

Non-patent Literature 4: Stark G R, Methods of Enzymology, 47: 129-132,1977.

Non-patent Literature 5: Nakagawa et al., J. Am. Chem. Soc., 116:5513-5514, 1994.

Non-patent Literature 6: Sola et al., J. Chem. Soc. Chem. Commun.,1786-1788, 1993.

Non-patent Literature 7: Pascal et al., Tetrahedron Letters, Vol. 35,No. 34: 6291-6294, 1994.

SUMMARY OF INVENTION Problem to be Solved by the Invention

In the peptide thioesterification shown in the above background art, thepeptide capable of being thioesterified is limited to the peptide chainsynthesized in the solid phase and the peptide chain to be targeted bythe protein splicing. This is because any of these methods require thenon-native amino acid derivative, the linker and the particularthree-dimensional structure, etc.

Thus, it is an object of the present invention to provide a novelprocess for chemically converting a polypeptide chain into a peptidethioester.

Means for Solving Problems

The present inventors considered that a process for selectivelyactivating a peptide C-terminal by targeting a native amino acid residuein a peptide sequence is required. In such a process, the peptide chainin any peptide obtained by any method such as biosynthesis can beselectively activated and thioesterified.

Thus, the present inventors focused on a cysteine residue that is aspecial sulfur-containing amino acid among the native amino acids. Andthe present inventors have found that as shown in the following figure,a —C(═X)—R₁ group is introduced into a thiol group of the cysteineresidue, and a compound having a leaving group represented by—NH—C(═Y)NHR₃ is reacted therewith in an organic solvent to add the—NH—C(═Y)NHR₃ group to a carboxyl group of a peptide bond on anN-terminal-side of the cysteine residue, whereby the peptide bond iscleaved and a peptide fragment on a C-terminal-side is cut off. Further,the present inventors have found that the resulting peptide chain can beconverted into a peptide thioester by an exchange reaction with thiol inwhich a thiol compound is reacted with the peptide chain to which the—NH—C(═Y)NHR₃ group has been added in a buffer solution, therebyallowing the thiol group of the thiol compound to be bound to carbonylcarbon to which the —NH—C(═Y)NHR₃ group has been bound and eliminatingthe —NH—C(═Y)NHR₃ group.

As one example of the above, specifically, a thionoformate group wasfirst introduced into the thiol group of the cysteine residue. And, apeptide chain in which N-acetylguanidido has been added to itsC-terminal was obtained by reacting N-acetylguanidine with thisthionoformate group in the organic solvent to cause the cleavage of thepeptide chain on the N-terminal-side of the cysteine residue. Further,this N-acetylguanidido-added peptide chain was reacted with thiol R₄—SHin the buffer solution to convert into the peptide thioester.

The present inventors have also found that the N-acetylguanidido-addedpeptide chain and the peptide thioester obtained above can be used inthe NCL method.

That is, the present invention specifically provides the following [1]to [14].

-   [1]

A process for producing a peptide thioester, comprising the followingsteps (a) to (c):

(a) a step of producing a first intermediate by reacting a compound Arepresented by the following formula (I) with a thiol group of acysteine residue in a peptide chain having the cysteine residue toeliminate R₂:

wherein X is a sulfur atom or an oxygen atom, R₁ and R₂ are leavinggroups;

(b) a step of reacting a compound B represented by the following formula(II) with the first intermediate in an organic solvent to add a—NH—C(═Y)NHR₃ group to a carboxyl group forming a peptide bond with anamino acid adjacent to an N-terminal-side of the cysteine residue, andcleaving the peptide bond, thereby obtaining a peptide fragment from theN-terminal-side closer to the N-terminal side than the cleaved peptidebond as a second intermediate:

wherein Y is an oxygen atom, a sulfur atom or an NH group and R₃ is ahydrogen atom, an acyl group or an alkoxycarbonyl group; and

(c) a step of thioesterifying a C-terminal of the second intermediate byreacting the second intermediate with thiol to exchange the—NH—C(═Y)NHR₃ group for the thiol group at the C-terminal.

-   [2]

The process according to above [1], wherein X is the sulfur atom.

-   [3]

The process according to above [1] or [2], wherein R₁ is a —O—C₆ arylgroup.

-   [4]

The process according to any of above [1] to [3], wherein R₂ is ahalogen atom or a substituted or unsubstituted —S—C₆₋₁₀ aryl group.

-   [5]

The process according to any of above [1] to [4], wherein Y is an NHgroup.

-   [6]

The process according to any of above [1] to [5], wherein R₃ is anacetyl group.

-   [7]

The process according to any of above [1] to [6], wherein the thiol isthiol represented by the following formula (III) in the step (c):R₄—SH  (Formula III)wherein R₄ is any one group selected from a substituted or unsubstitutedbenzyl group, a substituted or unsubstituted aryl group, and asubstituted or unsubstituted alkyl group.

-   [8]

The process according to any of above [1] to [7], wherein the peptidechain is a recombinant protein.

-   [9]

The process according to any of above [1] to [8], wherein the peptidechain is a recombinant protein comprising a tag for purification.

-   [10]

A process for producing a polypeptide comprising a step of binding thepeptide thioester obtained by the process according to any of above [1]to [9] to a peptide chain having cysteine at a N-terminal by a ligationmethod.

-   [11]

A process for producing a second intermediate used for the process forproducing the peptide thioester according to any of [1] to [9] above,comprising:

(a) a step of producing a first intermediate by reacting a compound Arepresented by the following formula (I) with a thiol group of acysteine residue in a peptide chain having the cysteine residue toeliminate R₂:

wherein X is a sulfur atom or an oxygen atom, R₁ and R₂ are leavinggroups; or

(b) a step of reacting a compound B represented by the following formula(II) with the first intermediate in an organic solvent to add a—NH—C(═Y)NHR₃ group to a carboxyl group forming a peptide bond betweenthe cycteine residue and an amino acid adjacent to an N-terminal-side ofthe cysteine residue, and cleaving the peptide bond, thereby obtaining apeptide fragment from the N-terminal-side closer to the N-terminal-sidethan the cleaved peptide bond as a second intermediate:

wherein Y is an oxygen atom, a sulfur atom or an NH group and R₃ is ahydrogen atom, an acyl group or an alkoxycarbonyl group.[12]

A peptide chain having a —NH—C(═Y) NHR₃ group at a C-terminal, wherein Yis an oxygen atom or an NH group and R₃ is a hydrogen atom, an acylgroup or an alkoxycarbonyl group.

[13]

A process for producing a polypeptide comprising a step of binding thepeptide chain having the —NH—C(═Y)NHR₃ group at the C-terminal accordingto above [12] to a peptide chain having cysteine at an N-terminal by aligation method.

[14]

A process for removing a tag for purification added to a C-terminal-sideof a recombinant protein, comprising the following steps (a) to (c):

(a) a step of producing a first intermediate by reacting a compound Arepresented by the following formula (I) with a thiol group of acysteine residue in the recombinant protein containing the tag forpurification on the C-terminal-side to eliminate R₂:

wherein X is a sulfur atom or an oxygen atom, R₁ and R₂ are leavinggroups;

(b) a step of reacting a compound B represented by the following formula(II) with the first intermediate in an organic solvent to add a—NH—C(═Y)NHR₃ group to a carboxyl group forming a peptide bond betweenthe cysteine residue and an amino acid adjacent to an N-terminal-side ofthe cysteine residue, and cleaving the peptide bond, thereby obtaining apeptide fragment from the N-terminal-side closer to the N-terminal-sidethan the cleaved peptide bond as a second intermediate:

wherein Y is an oxygen atom, a sulfur atom or an NH group and R₃ is ahydrogen atom, an acyl group or an alkoxycarbonyl group; and

(c) a step of thioesterifying a C-terminal of the second intermediate byreacting the second intermediate with thiol to exchange the—NH—C(═Y)NHR₃ group for the thiol group at the C-terminal.

Effects of the Invention

According to the present invention, a novel process for chemicallyconverting the polypeptide chain into the peptide thioester has beenprovided.

In the process of the present invention, the peptide chain not havingthe non-native amino acid derivative, the linker and the particularthree-dimensional structure, etc., required for the conventionalthioesterification methods can be thioesterified. Therefore, even thelong chain polypeptide fragment obtained by the biosynthesis, etc., canbe thioesterified easily.

Further, by combining the process of the present invention with theconventional peptide synthesis method, the long chain polypeptidepartially having the peptide modification, which was so far difficult tobe synthesized can be produced easily and simply by making a fragment ofthe portion having no modification using the biosynthesis method bywhich the long chain is relatively easily synthesized, making a fragmentof the portion having the modification using the solid-phase synthesismethod, and ligating them.

More specifically, the longer sugar chain peptide can be produced easilyand simply by chemically synthesizing a fragment alone containing aminoacids to which a native binding form of the sugar chain has been addedwhen the modification is performed with the sugar chain, preparing theother portion by biosynthesis and thioesterifying it by the process ofthe present invention, and ligating them.

The method of subsequently adding the sugar chain and the like to thepeptide chain via the linker is also known publicly, and this can alsosubsequently add the sugar chain to the biosynthesized long chainpeptide. However, this sugar chain binding method via the linker bindsthe sugar chain and the like by utilizing the particular amino acid andits structure. Therefore, for example, when multiple sites capable ofbinding the sugar chain are present in the peptide, the sugar chain canbe added more easily and simply in the site-specific manner comparedwith the conventional methods, by cutting out a peptide fragmentcontaining the desired binding site alone from a long chain peptideafter obtaining the long chain peptide by biosynthesizing, adding thesugar chain thereto, thioesterifying the sugar chain-added peptidefragment using the thioesterification process of the present invention,and ligating again to the remaining portion.

Further, in the biosynthesis, even if the full length protein isnormally expressed, its peptide fragment can be wrongly recognized,degraded or not expressed normally in the cell. It is also possible thatafter synthesizing the full length protein, its fragment alone to bemodified is cut out, the necessary treatment such as modification isgiven thereto, the modified peptide fragment is thioesterified using theprocess of the present invention, and the thioesterified fragment isagain ligated to the remaining portion to yield the desired modifiedprotein.

As described above, the peptide thioesterification process of thepresent invention is generally useful for synthesis of the proteins.

DETAILED DESCRIPTION OF PREFERRED EMBODIMENTS

Suitable embodiments of the present invention will be described below.

The present invention provides a novel process for producing the peptidethioester, comprising the following steps (a) to (c):

(a) a step of producing a first intermediate by reacting a compound Arepresented by the following formula (I) with a thiol group of acysteine residue in a peptide chain having the cysteine residue toeliminate R₂:

wherein X is a sulfur atom or an oxygen atom, R₁ and R₂ are leavinggroups;

(b) a step of reacting a compound B represented by the following formula(II) with the first intermediate in an organic solvent to add a—NH—C(═Y)NHR₃ group to a carboxyl group forming a peptide bond betweenthe cysteine residue and an amino acid adjacent to an N-terminal-side ofthe cysteine residue, and cleaving the peptide bond, thereby obtaining apeptide fragment from the N-terminal-side closer to the N-terminal-sidethan the cleaved peptide bond as a second intermediate:

wherein Y is an oxygen atom, a sulfur atom or an NH group and R₃ is ahydrogen atom, an acyl group or an alkoxycarbonyl group; and

(c) a step of thioesterifying a C-terminal of the second intermediate byreacting the second intermediate with thiol to exchange the—NH—C(═Y)NHR₃ group for the thiol group at the C-terminal.

In the present invention, the “peptide” is not particularly limited aslong as two or more amino acids are bound via amide bond(s), andincludes publicly known peptides, novel peptides and modified peptides.Those commonly referred to as the protein are included in the peptidesin the present invention. Also in the present invention, the“polypeptide” is included in the peptides. The peptide chain used forthe process of the present invention may be the native protein or thepeptide chain obtained by methods such as the biosynthesis, the chemicalsynthesis or the cell-free synthesis.

In the present invention, the “modified peptide” includes naturalvariants of the peptides, post-translational modified peptides, orartificially modified compounds. Such an modification includes, forexample, alkylation, acylation (e.g., acetylation), amidation (e.g.,amidation of C-terminal of peptide), carboxylation, ester formation,disulfide bond formation, glycosylation, lipidation, phosphorylation,hydroxylation, binding of labeled component, etc., in one or more aminoacid residues in the peptide.

In the present invention, the “amino acid” is used in its broadestcontext, and includes not only native amino acids such as serine (Ser),asparagine (Asn), valine (Val), leucine (Leu), isoleucine (Ile), alanine(Ala), tyrosine (Tyr), glycine (Gly), lysine (Lys), arginine (Arg),histidine (His), aspartic acid (Asp), glutamic acid (Glu), glutamine(Gln), threonine (Thr), cysteine (Cys), methionine (Met), phenylalanine(Phe), tryptophan (Trp) and praline (Pro), but also non-native aminoacids such as amino acid variants and derivatives. Those skilled in theart will understand that the amino acids in the present inventioninclude, for example, L-amino acids; D-amino acids, chemically modifiedamino acids such as amino acid variants and derivatives; amino acidssuch as norleucine, β-alanine and ornithine that are not constitutivematerials for the proteins in vivo; and compounds chemically synthesizedto have amino acid properties known to those skilled in the art, etc.,in consideration of this broad definition.

In the present invention, the peptide chain to be thioesterified is notparticularly limited as long as the peptide chain contains the cysteineresidue. For example, an origin, a synthesis method, a size and the likeof the peptide chain are not particularly limited. The peptide chain mayalso have the modification and a protective group.

The number of cysteine residues contained in the peptide chain used inthe present invention is not particularly limited, and the peptide chainis cleaved by targeting the cysteine residues. Therefore, it isnecessary to design a basic skeleton of the finally synthesized proteindepending on the sites having the cysteine residue, and those skilled inthe art can easily design such a basic skeleton. The cysteine residuesother than the desired cysteine residues may be protected withprotective groups in advance so that the thioesterification is performedat positions of only the desired cysteine residues and the remainingcysteine residues are not affected by the reaction in the peptide chaincontaining the multiple cysteine residues. Examples of such a protectivegroup include an Acm group.

The peptide chain used in the present invention may have a fat-solubleprotective group on the N-terminal-side. Preferable protective groupscan include, but are not limited to acyl groups such as an acetyl (Ac)group, carbonyl-containing groups such as a t-butyloxycarbonyl (Boc)group, a 9-fluorenylmethoxycarbonyl (Fmoc) group and an allyloxycarbonyl(Alloc) group, and an allyl group and a benzyl group.

The peptide chain used for the process of the present invention may be anative protein or the peptide chain obtained by methods such as thebiosynthesis, the chemical synthesis or the cell-free synthesis, and ispreferably a recombinant protein expressed in a bacterial cell or acell. The recombinant protein may be those having the same peptidesequence as in the native protein or those having the peptide sequencehaving the modification such as a tag for mutation or purification aslong as the protein is expressed artificially in the bacterial cell orthe cell.

The recombinant protein used in the present invention can be prepared bythe method known to those skilled in the art. For example, therecombinant protein can be expressed by introducing an objective geneinto a recombinant vector. The recombinant vector used in the presentinvention may be those capable of transforming a host cell, and aplasmid for Escherichia coli, a plasmid for Bacillus subtilis, a plasmidfor yeast, and animal virus vectors such as retrovirus, vaccinia virusand baculovirus are used. These preferably have a reguratory sequencesuch as a promoter capable of appropriately expressing the protein inthe host cell. Moreover, the host cell may be those capable ofexpressing a foreign gene in the recombinant vector, and generallyEscherichia coli, Bacillus subtilis, yeast, insect cells and animalcells are used.

The method ordinarily used in general may be used as the method oftransfecting the recombinant vector into the host cell. For example, acalcium chloride method and an electroporation method in the case ofEscherichia coli and a lithium chloride method and the electroporationmethod in the case of the yeast can be utilized. Transformation of theanimal cell can be performed using a physical method such as theelectroporation method, a chemical method such as a liposome method anda calcium phosphate method, or a viral vector such as retrovirus. Aculture condition of the host cell that is a transformant may beselected in consideration of nutritional and physiological properties ofthe host cell.

It is preferable that the peptide used in the present invention ispurified. The peptide can be purified by an ordinary purificationmethod. For example, in the case of a recombinant protein, a bacterialcell or a cell expressing the recombinant protein used in the presentinvention is cultured, subsequently the bacterial cell or the cell iscollected by a known method, then suspended in an appropriate buffersolution, disrupted by sonication, lysozyme and/or freezing and thawing,and then a crude extract solution of a peptide is prepared bycentrifugation or filtration. A protein denaturing agent such as ureaand guanidine hydrochloride, and a surfactant such as Triton X-100™ maybe contained in the buffer solution. The peptide contained in theextract solution or the culture supernatant obtained as above can bepurified by the known purification method. For example, the peptide canbe isolated and purified by appropriately selecting and combiningaffinity chromatography, ion exchange chromatography, filter,ultrafiltration, gel filtration, electrophoresis, salting out, dialysis,and the like.

A tag for the purification can be incorporated into the expressionvector in order to make the purification of the recombinant proteineasy. Examples of the tag for the purification include, for example, anHis tag, a GST tag, a Myc tag, a FLAG tag, and a maltose-binding protein(MBP). In the present invention, the N-terminal-side from Cys arrangedwithin the peptide chain is thioesterified, thus the tag for thepurification is added to the C-terminal-side of the peptide and thepeptide after the purification is thioesterified, whereby theC-terminal-side from Cys in the peptide chain which includes the tag iscut off and the peptide thioester can be obtained efficiently. Byarranging Cys to the desired position on the peptide chain, it is alsopossible to use the process of the present invention for removal of thetag on the C-terminal-side.

Therefore, the process for removing the tag for the purification addedto the C-terminal of the recombinant protein by using the process forproducing the peptide thioester of the present invention is alsoincluded in the present invention.

In the present invention, the “peptide thioester” (hereinafter sometimesalso simply described as the “thioester”) refers to the peptide having acarboxythioester moiety (—C═O—SR) at the C-terminal. The peptidethioester used in the present invention is not particularly limited aslong as the thioester can cause the exchange reaction with other thiolgroups. R group includes for example, groups exemplified in R₄ below.

In the process of the present invention, first (a) a step of reacting acompound A with a thiol group of a cysteine residue in a peptide chainhaving the cysteine residue for producing the first intermediate isperformed.

In the present invention, the compound A is represented by the followingformula (T).

In the formula, X is a sulfur atom or an oxygen atom, and preferably thesulfur atom.

R₁ and R₂ are not particularly limited as long as they have lowernucleophilicity than an atom or an atomic group to be substituted andhave a function to be eliminated under a reaction condition of thefollowing step (a) as leaving groups, and it is preferable that R₁ andR₂ are different leaving groups from each other. Examples of R₁ and R₂include specifically halogen atoms, substituted or unsubstituted—O-alkyl groups, substituted or unsubstituted —O-alkenyl groups,substituted or unsubstituted —O-alkynyl groups, substituted orunsubstituted —O-aryl groups, substituted or unsubstituted —O-heteroarylgroups, substituted or unsubstituted —S-alkyl groups, substituted orunsubstituted —S-alkenyl groups, substituted or unsubstituted —S-alkynylgroups, substituted or unsubstituted —S-aryl groups, or substituted orunsubstituted —S-heteroaryl groups. More preferably, examples of R₁ andR₂ include combinations of R₁ that is the leaving group selected fromthe group consisting of substituted or unsubstituted —O—C₅₋₁₀ arylgroups and substituted or unsubstituted —S—C₁₋₈ alkyl groups and R₂ thatis the leaving group selected from the group consisting of halogenatoms, substituted or unsubstituted alkyl groups and substituted orunsubstituted —S—C₆₋₁₀ aryl groups.

In the present invention, the “alkyl group” is a monovalent groupderived from aliphatic hydrocarbon by removing any one hydrogen atom,and has a subset of hydrocarbyl or hydrocarbon containing hydrogen andcarbon atoms. The alkyl group includes a straight chain or branchedchain structure. The alkyl group of the present invention preferablyincludes the alkyl groups having 1 to 8 carbon atoms. “C₁₋₈ alkyl group”indicates the alkyl group having 1 to 8 carbon atoms, and specificexamples thereof include methyl, ethyl, propyl, butyl, pentyl, hexyl,heptyl and octyl groups.

In the present invention, the “alkenyl group” is a monovalent grouphaving at least one double bond. Geometrical forms of the double bondscan take Entgegen (E), Zusammen (Z), cis or trans configurationsdepending on the configuration of the double bonds and substituents. Thealkenyl group includes the straight chain or branched chain form. Thealkenyl group of the present invention preferably includes the alkenylgroups having 2 to 8 carbon atoms. “C₂₋₈ alkenyl group” indicates thealkenyl group having 2 to 8 carbon atoms, and specific examples thereofinclude vinyl, allyl, propenyl, butenyl, pentenyl, hexenyl, heptenyl andoctenyl groups.

In the present invention, the “alkynyl group” is a monovalent grouphaving at least one triple bond. The alkynyl group includes straightchain or branched chain alkynyl groups. The alkynyl group of the presentinvention preferably includes the alkynyl groups having 2 to 8 carbonatoms. “C₂₋₈ alkynyl group” indicates the alkynyl group having 2 to 8carbon atoms, and specific examples thereof include ethynyl, 1-propynyl,2-propynyl, butynyl, pentynyl, hexynyl, heptynyl, and octynyl groups.

In the present invention, the “aryl group” means an aromatic hydrocarbonring group. The aryl group of the present invention preferably includesthe aryl groups having 6 to 10 carbon atoms “C₆₋₁₀ aryl group” indicatesthe aryl group having 6 to 10 carbon atoms, and specific examplesthereof include phenyl, 1-naphthyl and 2-naphthyl groups.

In the present invention, the “heteroaryl group” means a monovalent orbivalent group derived from a heteroaryl ring by removing one or twohydrogen atoms at any position(s). In the present invention, the“heteroaryl ring” means an aromatic ring having one or multipleheteroatoms in atoms composing the ring, and is preferably 5 to 9membered rings. The ring may be a monocyclic or bicyclic heteroarylgroup obtained by being fused with a benzene ring or a monocyclicheteroaryl ring. Specific examples thereof include furanyl, thiophenyl,pyrrolyl, benzofuranyl, benzothiophenyl, indolyl, pyridyl, and quinolylgroups.

Types, numbers and substituted positions of substituents that theaforementioned leaving groups have are not particularly limited, andexamples of the substituents include alkyl, alkenyl, alkoxy, aryl,formyl, carbonyl, carboxyl, alkylcarboxyl, alkoxycarbonyl, halogen,sulfonyl or nitro groups.

The compound A of the present invention includes more specifically thefollowings.

can be reacted with MPAA ((4-carboxymethyl)thiophenol) to produce thefollowing thionoformate reagent, which can also be used:

The first intermediate in which the —C(═X)—R₁ group is bound to the SHgroup in the cysteine residue as shown in the following figure can beobtained by reacting the compound A of the present invention with thecysteine residue in the peptide.

In the present invention, the step (a) is preferably performed under anacidic condition, particularly at pH 3 to 5. The reaction is preferablyperformed in a mixed solvent of buffer solution and acetonitrile at 0 to50° C., preferably 15 to 25° C. for about 0.1 to 3 hours, preferably 10minutes to one hour, but is not limited thereto.

Then, in the present invention, the step (b) is performed, in which thepeptide fragment on the N-terminal-side closer to the N-terminal-sidethan the cleaved peptide bond is obtained as the second intermediate byreacting the compound B with the first intermediate in the organicsolvent to add the —NH—C(═Y)NHR₃ group to the carboxyl group forming thepeptide bond with the amino acid adjacent to the N-terminal-side of thecysteine residue, and cleaving the peptide bond.

In the present invention, the compound B is represented by the followingformula (II).

In the formula, Y is an oxygen atom, an NH group or a sulfur atom, andR₃ is a hydrogen atom, an acyl group, or an alkoxycarbonyl group.

In the present invention, the “acyl group” means an atomic groupobtained by removing an OH group from a carboxyl group of a carboxylicacid. The acyl group of the present invention preferably includes theacyl groups having 1 to 4 carbon atoms, and specific examples thereofinclude acetyl, propionyl and butyroyl groups.

In the present invention, the “alkoxy group” means an oxy group bound tothe “alkyl group.” The alkoxy group of the present invention may bestraight chain or branched chain. The alkoxy group of the presentinvention preferably includes the straight chain alkoxy groups having 1to 14 carbon atoms and the branched chain alkoxy groups having 3 to 14carbon atoms. Specifically, for example, methoxy, ethoxy, n-propyloxy,isopropoxy, n-butoxy, 2-methyl-2-propyloxy, n-pentyloxy, and n-hexyloxygroups can be included.

Also, “C_(2-n) alkoxycarbonyl group” means the carbonyl group having aC_(1-(n-1)) alkoxy group. The alkoxycarbonyl group of the presentinvention preferably includes the alkoxycarbonyl groups having 2 to 15carbon atoms. Specifically, for example, methoxycarbonyl,ethoxycarbonyl, n-propyloxycarbonyl, isopropoxycarbonyl,n-butoxycarbonyl, 2-methyl-2-propyloxycarbonyl, n-pentyloxycarbonyl, andn-hexyloxycarbonyl groups can be included.

The acyl group preferably includes the acetyl group. Also thealkoxycarbonyl group preferably includes a tert-butoxycarbonyl (Boc)group.

The compound B of the present invention includes more specifically thefollowing.

In the present invention, the step (b) is preferably performed in thepresence of the organic solvent. It is preferable that the organicsolvent is high in solubility and low in nucleophilicity. Such anorganic solvent can include, for example, DMSO, DMF and dioxane. Thereaction is preferably performed at 0 to 50° C., preferably 15 to 25° C.for about 1 to 24 hours, preferably 5 to 10 hours, but is not limitedthereto.

The peptide chain is cleaved on the N-terminal-side of the cysteineresidue as shown in the following figure by adding the —NH—C(═Y)NHR₃group to the carboxyl group forming the peptide bond between thecysteine residue and the amino acid adjacent to the N-terminal-side ofthe cysteine residue.

When the peptide has the amino group in its side chain, a fat-solubleprotective group may be introduced to the amino group in the side chainbefore performing the step (b) of the present invention. The fat-solubleprotective group can include, but is not limited to, protective groupssuch as carbonyl-containing groups such as a 9-fluorenylmethoxycarbonyl(Fmoc) group, a t-butyloxycarbonyl (Boc) group and an allyloxycarbonyl(Alloc) group, acyl groups such as an acetyl (Ac) group, and an allylgroup and a benzyl group.

In order to introduce the fat-soluble protective group, for example, theFmoc group can be introduced by adding 9-fluorenylmethyl-N-succinimidylcarbonate and sodium hydrogen carbonate and reacting them. The reactionis preferably performed at 0 to 50° C. preferably at room temperaturefor about 1 to 5 hours, but is not limited thereto.

The peptide fragment on the N-terminal-side closer to theN-terminal-side than the cleaved site of the cleaved peptide chain canbe obtained as the second intermediate by the following formula (I) inthe step (b).

The process for producing the peptide thioester of the present inventionfurther includes the step (c) of thioesterifying the C-terminal of thesecond intermediate by reacting thiol with the second intermediate toexchange the —NH—C(═Y)NHR₃ group at the C-terminal for the thiol group.

The second intermediate used for the step (c) may be isolated or neednot be isolated after the step (b).

In preferable embodiments, thiol represented by the following formula(III):R₄—SH  (Formula III)is used in the step (c).

R₄ is not particularly limited as long as it does not inhibit the thiolexchange reaction and becomes the leaving group in a substitutionreaction on carbonyl carbon. Preferably, R₄ is any one group selectedfrom substituted or unsubstituted benzyl groups, substituted orunsubstituted aryl groups and substituted or unsubstituted alkyl groups.More preferably, R₄ is any one group selected from the substituted orunsubstituted benzyl groups, substituted or unsubstituted C₆₋₁₀ arylgroups and substituted or unsubstituted C₁₋₈ alkyl groups. Morespecifically, R₄ can be selected from benzyl type leaving groups such asbenzylmercaptan, aryl type leaving groups such as thiophenol and4-(carboxymethyl)thiophenol, alkyl type leaving groups such as a2-mercaptoethanesulfonic acid group and 3-mercaptopropionate amide, etc.The type, the number and the substituted position of the substituentsthat these leaving groups have are not particularly limited.

The second intermediate is completely converted into the thioester asthe following figure by performing the step (c).

The peptide thioester obtained as in the above can be ligated to apeptide (or a modified peptide) which contains an amino acid residuehaving the —SH group at the N-terminal among the peptides or themodified peptides by using the ligation method. Therefore, the presentinvention also provides a process for producing a polypeptide comprisinga step of binding the peptide thioester obtained by the process of thepresent invention to the peptide chain having cysteine at the N-terminalby the ligation method.

It is also possible to use the second intermediate obtained in the step(b) in place of the above peptide thioester for the ligation method.

In the present invention, the “ligation method” includes not only thenative chemical ligation method (NCL method) described in PatentLiterature 1, but also the cases of applying the native chemicalligation method to the peptides containing the non-native amino acid andamino acid derivative (e.g., threonine derivative A, protectedmethionine, sugar chain-added amino acids, etc.). The peptide having thenative amide bond (peptide bond) at the ligated site can be produced bythe ligation method.

The ligation using the ligation method can be performed in any cases ofbetween the peptide and the peptide, between the peptide and themodified peptide, and between the modified peptide and the modifiedpeptide.

The terms used herein are used for describing particular aspects and arenot intended to limit the present invention.

The term “comprising” (also, “containing” and “including”) used hereinintends that the described respects (members, steps, elements andnumerals, etc.) are present except the cases to be understood obviouslydifferent in context, and it is not excluded that the respects (members,steps, elements and numerals, etc.) other than these are present.

Unless otherwise defined differently, all of the terms (includingtechnical terms and scientific terms) used herein have the same meaningsas those understood widely by those skilled in the art to which thepresent invention belongs. The terms used herein should be construed tohave the meanings coherent to the meanings in this specification and therelated technical field unless a different definition is otherwisemanifested, and should not be construed in idealized or unduly formalmeanings.

The aspects of the present invention are sometimes described withreference to the schematic view. When described in the schematic view,the embodiment is sometimes expressed in a exaggerated manner in orderto describe it clearly.

The terms such as first and second are used to express various elements,but it is understood that these elements are not to be limited to thoseterms. These terms are used only for distinguishing one element from theother element, and without departing from the scope of the presentinvention, it is possible that the first element is written as thesecond element, as well as the second element is written as the firstelement.

The present invention will be described in more detail with reference tothe following Examples. However, the present invention can be embodiedby various aspects, and is not to be construed to be limited to Examplesdescribed here.

EXAMPLES Example 1 Introduction of Thionoformate Group

(Synthesis of MPAA Phenyl Thionoformate)

MPAA ((4-carboxymethyl)thiophenol) (98 mg, 0.583 mmol) and phenylchlorothionoformate (103 μL, 0.76 mmol) were dissolved indichloromethane (400 μL), and the mixture was stirred at roomtemperature for one hour. After one hour, the reaction solution wasdiluted with 2.0 mL of chloroform, 1.0 mL of an aqueous solution ofsaturated sodium bicarbonate was added, and the mixture was extractedand washed with chloroform. A chloroform layer was washed with saturatedsaline, dried on magnesium sulfate, and then concentrated under reducedpressure to give a yellow clear residue in a syrup shape. This was thenused as a thionoformate reagent (MPAA phenyl thionoformate) (MW: 305.3,MS: no available data).

(Introduction of Thionoformate Group by MPAA Phenyl ThionoformateReagent)

A peptide (Ac-Val Try Ala Xaa Cys Gly-OH) (SEQ ID NO: 1), Xaa=Lys (SEQID NO: 2), Ser (SEQ ID NO: 3), Asp (SEQ ID NO: 4), Ala (SEQ ID NO: 5),Val (SEQ ID NO: 6), crude (mixture of Lys, Ser, Asp, Ala and Val), 6 mg)was dissolved in a buffer solution at pH 5.5 (1.0 mL of 0.2 MNa₂HPO₄ and6M Gn-HCl), and then a total amount of MPAA phenyl thionoformate (15 μL)dissolved in acetonitrile (230 μL) was added thereto. After one hour,the reaction solution was washed with Et₂O. The purification wasperformed by HPLC to yield an objective compound. The reaction wasquantitatively performed as a result of HPLC.

(Xaa=Lys, ESIMS calcd [M+H]⁺ 818.3, found [M+H]⁺ 818.4)

(Xaa=Ser, ESIMS calcd [M+H]⁺ 777.3, found [M+H]⁺ 777.3)

(Xaa=Asp, ESIMS calcd [M+H]⁺ 805.3, found [M+H]⁺ 805.3)

(Xaa=Ala, ESIMS calcd [M+H]⁺ 761.3, found [M+H]⁺ 761.3)

(Xaa-Val, ESIMS calcd [M+H]⁺ 789.3, found [M+H]⁺ - - -)

A peptide (Ac-Val Try Ala Xaa Cys Gly-OH) (SEQ ID NO: 1), Xaa=Ser (SEQID NO: 3), Phe (SEQ ID NO: 8), Leu (SEQ ID NO: 7), crude (mixture ofSer, Phe and Leu), 10 mg) was dissolved in the buffer solution at pH 5.0(2.0 mL of 0.2 M Na₂HPO₄ and 6 M Gn-HCl) and then MPAA phenylthionoformate (5 μL) dissolved in acetonitrile (700 μL) was addedthereto. After 1.5 hours, the reaction solution was washed with Et₂O.The purification was performed by HPLC to yield an objective compound.The reaction was quantitative as a result of HPLC.

(Xaa=Ser, ESIMS calcd [M+H]⁺ 777.3, found [M+H]⁺ 777.3)

(Xaa=Leu, ESIMS calcd [M+H]⁺ 803.4, found [M+H]⁺ 803.3)

(Xaa=Phe, ESIMS calcd [M+H]⁺ 837.4, found [M+H]⁺ 837.3)

The thionoformate group was introduced into the —SH group of cysteineregardless of the type of amino acid adjacent to the N-terminal-side ofthe cysteine.

Example 2 N-acetylguanidinylation Reaction

(Performed with Xaa=Ala, Leu, Phe, Ser and Lys)(Case of Xaa=Ala)

Ac-Val Tyr Ala Ala Cys(C(S)OPh)Gly-OH (SEQ ID NO: 9) (0.2 mg, 0.28 μmol)was dissolved in 250 mM N-acetylguanidine/DMSO solution (260 μL). Aftertwo hours, a compound was precipitated with and washed with Et₂O. Theobjective compound was purified by HPLC to yield objectiveN-acetylguanidido (Ac-Val Tyr Ala Ala-NHC(NH)NHAc (SEQ ID No: 10)(yield: 80%, calculated from HPLC area intensity).

(ESIMS calcd [M+H]⁺ 548.3, found [M+H]⁺ 548.4)

(Case of Xaa=Leu or Phe)

A mixture of Ac-Val Tyr Ala Leu Cys(C(S)OPh)Gly-OH (SEQ ID NO:11) (0.1mg, 0.12 μmol) and Ac-Val Tyr Ala Phe Cys (C(S)OPh) Gly-OH (SEQ ID NO:12) (0.1 mg, 0.12 μmol) was dissolved in 250 mM N-acetylguanidine/DMSOsolution (100 μL). After 4.5 hours, compounds were precipitated with andwashed with Et₂O. The objective compounds were purified by HPLC to yieldobjective N-acetylguanidido (Ac-Val Tyr Ala Leu-NHC(NH)NHAc (SEQ ID No:13) and Ac-Val Tyr Ala Phe-NHC(NH)NHAc (SEQ ID No: 14) (yield: 80%,calculated from HPLC area intensity).

(Xaa-Leu, ESIMS calcd [M+H]⁺ 590.3, found [M+H]⁺ 590.3)

(Xaa=Phe, ESIMS calcd [M+H]⁺ 624.3, found [M+H]⁺ 624.3)

(Case of Xaa=Ser)

Ac-Val Tyr Ala Ser Cys(C(S)OPh)Gly-OH (SEQ ID NO: 15) (0.2 mg, 0.26μmol) was dissolved in 250 mM N-acetylguanidine/DMSO solution (100 μL).After 3.5 hours, a compound was precipitated with and washed with Et₂O.The objective compound was purified by HPLC to yield objectiveN-acetylguanidido (Ac-Val Tyr Ala Ser-NHC(NH)NHAc (SEQ ID No: 16)(yield: 70%, from HPLC area intensity).

(Case of Xaa=Lys)

A peptide (Ac-Val Tyr Ala Lys Cys (C(S)OPh)Gly-OH (SEQ ID NO: 17) (0.1mg) was dissolved in DMSO (30 μL) containing Boc₂O (0.3 mg) andtriethylamine (0.14 μL). After 1.5 hours, the reaction solution wasprecipitated with and washed with Et₂O. The resulting residue wasdissolved in 250 mM N-acetylguanidine/DMSO solution (100 μL). After 2.5hours, the objective compound was purified by HPLC to yield objectiveN-acetylguanidido (Ac-Val Tyr Ala Lys(Boc)-NHC (NH)NHAc (SEQ ID No: 18)(yield: 70%, calculated from HPLC area intensity).

It was identified that the cysteine residue to which the thionoformatehad been added had the reactivity with guanidine to the peptide bond onthe N-terminal-side, regardless of the type of amino acid adjacent tothe N-terminal-side of the cysteine.

Example 3 Thioesterification of 24 aa Peptide

A peptide (H₂N-Leu Ile Cys(Acm)Asp Ser Arg Val Leu Glu Arg Tyr Leu LeuGlu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys Gly-OH (SEQ ID NO: 19),crude (not purified)), appropriate amount (estimated about 1 mg) wasdissolved in the buffer solution at pH 5.0 (300 μL of 0.2 M Na₂HPO₄ and6 M Gn-HCl), and then the total amount of MPAA phenyl thionoformate (1μL) dissolved in acetonitrile (100 μL) was added thereto. After 50minutes, the reaction solution was washed with Et₂O. The purificationwas performed by HPLC to yield an objective compound (H₂N-Leu IleCys(Acm)Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys Glu Ala GlnAsn Ile Thr Thr Gly Cys(C(S)OPh)Gly-OH (SEQ ID NO: 20)). Cys at position3 was previously protected with Acm not to be affected with thethionoformate reagent.

(ESIMS calcd[M+2H]²⁺1553.8, [M+3H]³⁺ 1035.8, found [M+2H]²⁺ 1552.9,[M+3H]³⁺ 1035.7)

The peptide (H₂N-Leu Ile Cys(Acm)Asp Ser Arg Val Leu Gln Arg Tyr Leu LeuGlu Ala Lys Glu Ala Glu Asn Ile Thr Thr Gly Cys(C(S)OPh)Gly-OH (SEQ IDNO: 20), ca. 0.3 mg) was dissolved in DMSO (20 μL) containing Boc₂O (0.4mg) and triethylamine (0.03 μL). After 1.5 hours, the reaction solutionwas precipitated with and washed with Et₂O. The resulting residue wasdissolved in 250 mM N-acetylguanidine/DMSO solution (50 μL). After 2.5hours, the objective compound was purified by HPLC to yield objectiveN-acetylguanidido (BocHN-Leu Ile Cys(Acm)Asp Ser Arg Val Leu Glu Arg TyrLeu Leu Glu Ala Lys(Boc)Glu Ala Glu Asn Ile Thr Thr Gly-NHC(NH)NHAc (SEQID NO: 21)).

(ESIMS calcd [M+2H]²⁺ 1546.8, [M+3H]³⁺ 1031.5, found [M+2H]²⁺1547.0,[M+3H]³⁺ 1031.4)

The 24 aa peptide (BocHN-Leu Ile Cys(Acm)Asp Ser Arg Val Leu Glu Arg TyrLeu Leu Glu Ala Lys(Boc)Glu Ala Gln Asn Ile Thr Thr Gly-NHC(NH)NHAc (SEQID NO: 21), ca. 0.1 mg>) was dissolved in a buffer solution at pH 7.05(0.2 Mphosphoric acid, 6 M guanidine, 50 μL) containing MESNa (sodium2-sulfanylethanesulfonate) (1 mg, 2% v/v). After 3.5 hours, theobjective compound was purified by HPLC to yield a thioester (BocHN-LeuIle Cys(Acm)Asp Ser Arg Val Leu Glu Arg Tyr Leu Leu Glu Ala Lys(Boc)GluAla Glu Asn Ile Thr Thr Gly-SCH₂CH₂SO₃ (SEQ ID NO: 22)) (unknown yield,about 70% on HPLC).

(ESIMS calcd [M+2H]²⁺ 1567.3, found [M+2H]²⁺ 1566.8)

Example 4 Introduction of Thionoformate Group by ChlorothionoformateReagent

The peptide (Ac-Val Tyr Ala Ala Cys Gly-OH (SEQ ID NC: 5), 6 mg) wasdissolved in the buffer solution at pH 5.0 (961 μL of 0.2 M Na₂HPO₄ and6M Gn-HCl), and phenyl chlorothionoformate (6.5 μL) dissolved inacetonitrile (320 μl) was added thereto. After one hour, the reactionsolution was washed with Et₂O. The purification was performed by HPLC toyield an objective thionoformate-added peptide (SEQ ID NO: 9) (6.4 mg,88%). (Xaa=Ala, ESIMS calcd [M+H]⁺ 761.3, found [M+H]⁺ 761.3)

The peptide (Ac-Val Tyr Ala Leu Cys Gly-OH (SEQ ID NO: 7), 3.4 mg) wasdissolved in the buffer solution at pH 5.0 (510 μL of 0.2 μM Na₂HPO₄ and6 M Gn-HCl), and phenyl chlorothionoformate (3.5 μl) dissolved inacetonitrile (170 μL) was added thereto. After one hour, the reactionsolution was washed with Et₂O. The purification was performed by HPLC toyield an objective thionoformate-added peptide (SEQ ID NO: 11) (3.8 mg,92%). (Xaa=Leu, ESIMS calcd [M+H]⁺803.4, found [M+H]⁺803.3)

The peptide (Ac-Val Tyr Ala Phe Cys Gly-OH (SEQ ID NO: 8), 5.1 mg) wasdissolved in the buffer solution at pH 5.0 (729 μL of 0.2 M Na₂HPO₄ and6M Gn-HCl), and phenyl chlorothionoformate (5.0 μL) dissolved inacetonitrile (243 μl) was added thereto. After one hour, the reactionsolution was washed with Et₂O. The purification was performed by HPLC toyield an objective thionoformate-added peptide (SEQ ID NO: 12) (5.1 mg,84%). (Xaa-Phe, ESIMS calcd [M+H]⁺837.4, found [M+H]⁺837.3)

The peptide (Ac-Val Tyr Ala Ser Cys Gly-OH (SEQ ID NO: 3), 4.9 mg) wasdissolved in the buffer solution at pH 5.0 (766 μL of 0.2 M Na₂HPO₄ and6M Gn-HCl), and phenyl chlorothionoformate (5.2 μL) dissolved inacetonitrile (265 μL) was added thereto. After one hour, the reactionsolution was washed with Et₂O. The purification was performed by HPLC toyield an objective thionoformate-added peptide (SEQ ID NO: 15) (5.5 mg,92%). (Xaa=Ser, ESIMS calcd [M+H]⁺ 777.3, found [M+H]⁺ 777.3)

The peptide (Ac-Val Tyr Ala Lys Cys Gly-OH (SEQ ID NO: 2), 5.5 mg) wasdissolved in the buffer solution at pH 5.0 (810 μL of 0.2 M Na₂HPO₄ and6M Gn-HCl), and phenyl chlorothionoformate (5.5 μL) dissolved inacetonitrile (270 μL) was added thereto. After one hour, the reactionsolution was washed with Et₂O. The purification was performed by HPLC toyield an objective thionoformate-added peptide (SEQ ID NO: 17) (6.1 mg,94%). (Xaa=Lys, ESIMS calcd [M+H]⁺818.3, found [M+H]⁺818.4)

Using the chlorothionoformate reagent, the same thionoformate-addedpeptide chain as that obtained in Example 1 was obtained. Therefore, ithas been found that the peptide thioester can also be obtained from thepeptide chain in which the thionoformate group was introduced by thechlorothionoformate reagent by performing the N-acetylguanidido additionand then the thioesterification in the same manner as in the peptidechain in which the thionoformate group was introduced in the aboveExamples 1 and 3.

Industrial Applicability

According to the present invention, a novel process for chemicallyconverting the polypeptide chain into the peptide thioester wasprovided.

In the process of the present invention, the thioesterification ispossible in the peptide chain that does not have the non-native aminoacid derivative, the linker or the particular three dimensionalstructure, etc., required for the conventional thioesterificationmethod, and it is possible to easily thioesterify even in the long chainpolypeptide fragment obtained by the biosynthesis, etc. Therefore, thethioesterification process of the present invention can be generallyutilized for the synthesis of the proteins.

The invention claimed is:
 1. A process for removing a tag forpurification added to a C-terminal-side of a recombinant protein,comprising the following steps (a) to (c): (a) a step of producing afirst intermediate by reacting a compound A represented by the followingformula (I) with a thiol group of a cysteine residue to eliminate R₂ ina recombinant protein comprising a tag for purification at itsC-terminal:

wherein X is a sulfur atom or an oxygen atom, and R₁ and R₂ are leavinggroups; (b) step of reacting a compound B represented by the followingformula (II) with said first intermediate in an organic solvent to add a—NH—C(═Y)NHR₃ group to a carboxyl group forming a peptide bond betweenthe cysteine residue and an amino acid adjacent to an N-terminal-side ofsaid cysteine residue, and cleaving said peptide bond, thereby obtaininga peptide fragment from the N-terminal-side closer to theN-terminal-side than the cleaved peptide bond as a second intermediate:

wherein Y is an oxygen atom, a sulfur atom or an NH group and R₃ is ahydrogen atom, an acyl group or an alkoxycarbonyl group; and (c) a stepof thioesterifying a C-terminal of the second intermediate by reactingthiol with the second intermediate to exchange the —NH—C(═Y)NHR₃ groupat the C-terminal for the thiol group.
 2. The process according to claim1, wherein X is the sulfur atom.
 3. The process according to claim 1,wherein R₁ is a —O—C₆ aryl group.
 4. The process according to claim 2,wherein R₁ is a —O—C₆ aryl group.
 5. The process according to claim 1,wherein R₂ is a halogen atom, or a substituted or unsubstituted —S—C₆₋₁₀aryl group.
 6. The process according to claim 2, wherein R₂ is a halogenatom, or a substituted or unsubstituted —S—C₆₋₁₀ aryl group.
 7. Theprocess according to claim 3, wherein R₂ is a halogen atom, or asubstituted or unsubstituted —S—C₆₋₁₀ aryl group.
 8. The processaccording to claim 1, wherein Y is an NH group.
 9. The process accordingto claim 2, wherein Y is an NH group.
 10. The process according to claim3, wherein Y is an NH group.
 11. The process according to claim 5,wherein Y is an NH group.
 12. The process according to claim 1, whereinR₃ is an acetyl group.
 13. The process according to claim 2, wherein R₃is an acetyl group.
 14. The process according to claim 3, wherein R₃ isan acetyl group.
 15. The process according to claim 5, wherein R₃ is anacetyl group.
 16. The process according to claim 8, wherein R₃ is anacetyl group.
 17. The process according to claim 1, wherein the thiol inthe step (c) is thiol represented by the following formula (III):R₄—SH  (Formula III) wherein R₄ is any one selected from a substitutedor unsubstituted benzyl group, a substituted or unsubstituted arylgroup, and a substituted or unsubstituted alkyl group.
 18. The processaccording to claim 2, wherein the thiol in the step (c) is thiolrepresented by the following formula (III):R₄—SH  (Formula III) wherein R₄ is any one selected from a substitutedor unsubstituted benzyl group, a substituted or unsubstituted arylgroup, and a substituted or unsubstituted alkyl group.
 19. The processaccording to claim 3, wherein the thiol in the step (c) is thiolrepresented by the following formula (III):R₄—SH  (Formula III) wherein R₄ is any one selected from a substitutedor unsubstituted benzyl group, a substituted or unsubstituted arylgroup, and a substituted or unsubstituted alkyl group.
 20. The processaccording to claim 5, wherein the thiol in the step (c) is thiolrepresented by the following formula (III):R₄—SH  (Formula III) wherein R₄ is any one selected from a substitutedor unsubstituted benzyl group, a substituted or unsubstituted arylgroup, and a substituted or unsubstituted alkyl group.